Agents having a fibrinolytic activity and being derived from molds and a process of making and using same



June 1966 A. P. TRUANT ETAL 3, 6, 57

AGENTS HAVING A FIBRINOLYTIC ACTIVITY AND BEING DERIVED FROM MOLDS AND APROCESS OF MAKING AND USING SAME Filed Feb. 20, 1959 2 Sheets-Sheet lTij. l.

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AGENTS HAVING A FIBRINOLYTIC ACTIVITY AND BEING DERIVED FROM MOLDS AND APROCESS OF MAKING AND USING SAME Filed Feb. 20, 1959 2 Sheets-Sheet 2Mwszmem //v MICfO YS 3 S 6 7 8 9 I0 I! I2 13 145/6 52 4500 4000 am Z000I900 I700 I600 I400 I500 1200 1100 I000 900 'am no 6117 IN V EN TORS rcao P. T uA/vr Fflrz G. Noeosrycn MWMWOL AGE VT United States Patent 3256,157 AGENTS HAVING A FIBRINOLYTIC ACTIVITY AND BEING DERIVED FROMMOLDS AND A PROCESS OF MAKING AND USING SAME Aldo Peter Truant and FritzGeorge Norstrom, Worcester, Mass., assignors to Astra PharmaceuticalProducts, Inc., Worcester, Mass., a corporation of New York Filed Feb.20, 1959, Ser. No. 794,729 7 Claims. (Cl. l95--62) The present inventionrelates to agents having fibrinolytic activity and more particularly toagents having fibrinolytic activity and being derived from molds whichagents have a selective fibrinolytic activity at the site of a bloodclot without substantially affecting other blood and plasmaconstituents, and to a process of making and using same.

As is known, the clotting of blood will occur in the intact vascularsystem. If a clot does develop, the problem of thrombosis may arise andthe resulting thrombus may lead to infarction and, as a result thereof,to necrosis of the tissue supplied by the affected blood vessel. A bloodclot or embolus forced by the blood current from a thrombosed vesselinto a smaller one or formed in a coronary artery may cause obstructionof vital areas of the body and often death.

A number of agents have been used in therapy to prevent an extension ofthe blood clot or to cause its dissolution. Heparin or otheranticoagulants, for instance, have been administered for their propertyof prolonging the clotting time of blood which facilitates clotresolution and prevents extension of an existing intravascular clot.

Other agents, such as the enzyme streptokinase, have an activatingelfect on the precursor of the fibrinolytic enzyme profibrinolysin whichcauses cleavage of fibrin, i.e. the material comprising the blood clot,into soluble polypeptides. An agent of similar activity isstreptodornase. The enzyme trypsin has also been used for the lysis offibrin and plasma clots, likewise the enzyme plasmin obtained from humanblood.

These known agents, however, have a number of disadvantages.

(l) Heparin and coumarins do not directly alI'ect an existing blood clotand they do not limit the damage to the afiected vascular tissue.

(2) Trypsin, in doses insufiicient to induce clot lysis, causes anacceleration of the clotting mechanism and intravascular coagulation.There is no satisfactory means available to determine a priori the dosenecessary for fibrinolytic activity.

(3) Streptokinase, although not antigenic, possesses a pyrogenic action.

(4) Plasmin is expensive and has an antigenic action.

(5) Both trypsin and streptokinase measurably affect other clotting andplasma constituents. Therefore, these known agentshave found onlylimited application in therapy.

It is one object of the present invention to provide new and valuablefibrinolytic agents which difier in their chemical constitution fromthat of other known fibrinolytic agents, do not substantially affect theother blood and plasma constituents, and have substantially no antigenicand/ or pyrogenic activity.

, Another object of the present invention is to provide a startingmaterial for the preparation of such new and valuable fibrinolyticagents which material is readily available and permits large scaleproduction of such agents.

A further object of the present invention is to provide a simple andeffective process of producing such new and valuable fibrinolyticagents.

Still another object of the present invention is to .providesimple andelfective means of purifying and con- 3,256,157 Patented June 14, 1966ice centrating such fibrinolytic agents and to produce products with asurprisingly high degree of activity.

Still another object of the present invention is to provide a simple andefiective method of testing the starting material for its fibrinolyticactivity, of determiningthe activity of the intermediate and finalproducts obtained in the course of the manufacture of the newfibrinolytic agent, and of standardizing pharmaceutical preparations forclinical use containing such an agent.

Other objects of the present invention and advantageous features thereofwill become apparent as the description proceeds.

In principle, the present invention consists in isolating fibrinolyticagents from cultures of specific molds that are capable of producingsuch agents. a

Of a large number of molds, the following four strains which have provedto be valuable starting material for the purpose of the presentinvention were supplied for the isolation and purification of thefibrinolytic agents according to the present invention.

Two strains of the species Aspergillus oryzae, which are available tothe public from the Quartermaster Research and Engineering Center,United States Army, at Natick, Mass., under the depository numbers B-82iand B-1273, are capable of producing especially largeamounts of the newfibrinolytic agents. One strain of the species Aspergillus flavus, alsoavailable to the public from said Quartermaster Research and EngineeringCenter under the depository number B-4m, and one strain of the genusAbsidia, species Absidia coerulea, also available to the public fromsaid Quartermaster Research and Engineering Center under the depositorynumber D-10l,- have also proved to produce said new fibrinolytic agents.It is understood, of course, that although these specific strains haveproved to be especially suitable for the production of the newfibrinolytic agents, the present invention is not limited thereto. Anyother strain of molds which, when tested as will be describedhereinafter, shows selective vfibrinolytic activity, may also be used asstarting material.

The cultures investigated were transferred to solid Sabourauds medium.The Sabourauds dextrose medium contained Neopeptone g 10 Bacto-dextroseg 40 Bacto-agar g 15 Water, to cc-.. 1000 After cultivating the molds onsaid solid medium at 30 37 C. for five to ten days, the resultinginoculum was transferred to a liquid mediumprepared from Sucrose g 7.2

Dextrose g 3.6

MgSO (crystals) g 1.23 1 0 g 13.69

KNO3 g 2 and H 0, to cc 1000 Aliquots of cc. were transferred toErlenmeyers flasks of 250 cc. capacity. A sample of the Sabouraudculture, approximately 1 cm. in diameter, was transferred to each flask.Cultures were incubated at 29-30 C.

After a specific interval of time, the content of the flasks,

The precipitate is then dried in a vacuum at 37 C. According to theactivity of the precipitate amounts between 0.5 mg. and 10.0 mg. of thedried material are suspended in 1 cc. each of distilled :water, salinesolution, or phosphate buffer solution of a pH of 5.5, the total volumeof the suspension being one tenth of the volume of the originalfiltrate. The resulting suspensions or, respectively, solutions areassayed by two methods:

1) Fresh platelet-poor human or bovine plasma in a volume of 0.2 cc.which did not show retraction four hours after clotting, were mixed in atest tube with 0.2 cc. of saline solution, 0.2 cc. of the abovedescribed suspension or solution of the active agent, and 0.2 cc. ofthrombin solution containing 100 NIH units/ cc. After verifying thatclots had formed normally, the test tubes were observed for lysis hourlyfor 6 hours whereby they were kept in a water bath at 37 C. If, onaddition of 0.2 cc. of the suspension or solution of the precipitate, aclot does not occur, the suspension or solution must be further diluted.All molds which on cultivation on the above given culture medium yieldextracts showing clot lysing activity in the above given test wereselected for cultivation on a large scale and for production of the newfibrinolytic agents. It may be mentioned that this test is not Verysensitive. This is due to the pH-value of the mixture being about6.87.2. At such a pH-value the fibrinolytic activity of the active agentis considerably inhibited. Consequently, the concentration of the activeagent in the above described suspension or solution must be increased,if the test results are negative.

(2) The following second test method is more suitable for detectingfibrinolytic activity in molds andfor determining the activity offibrinolytic agents. According to this method modified Astrup-Muellertzfibrin plates are prepared by rapidly adding 1 cc. of bovine thrombinsolution containing 100 NIH units/ cc. to 10 cc. each of an 0.2% and a1% solution of bovine or human fibrinogen. After rapidly mixing saidsolutions, the mixture is poured into petri dishes. for minutes.Thereafter a drop of 0.2 cc. of the mold culture filtrate or,respectively, of the above described suspensions or solutions aredeposited on each plate by means of a pipette. After incubation in athermostat at 37 C. up to 180 minutes, the degree of lysis is judged bymeasuring the area of clearing and digestion of the fibrin plate.

It may be mentioned that this test method is more sensitive than theabove mentioned first test method. The reason for this is that thepH-value of the filtrate as well as of the suspensions or, respectively,solutions is at the optimum pH-value of activity of the fibrinolyticagent and that, therefore, activity can be detected much more easilythan with the first test method in which the filtrate or solutions orsuspensions are mixed with the test medium having a pH value of 6.8-7.2and, thus, are not at the optimum.

In the second method, although the pH-value of the plate is similar tothat of the test medium of the above mentioned first method, namely at6.8-7.2, the suspension or solution to be tested is not mixed with thetest medium but acts thereon at one point only, namely at the placewhere the drop is deposited. Therefore, the pH-value of the mold culturefiltrate which is between about 5.0 and 5.5, depending upon theconditions of cultivation, and the pH-value of the suspension orsolution which is at about 5 .5 are capable of exerting their activatingeffect and are not neutralized on deposition on the plates. A pH-valueof 5.5 has proved to be the optimum pH-value of activity of the'newfibrinolytic agents according to the present invention.

The following Table I shows the lytic activity of an alcoholicprecipitate of an active mold filtrate prepared as described above. 0.2cc. of the suspension having a pH of 5.5 were tested according to theabove mentioned second test method. The diameter size of clearing isdeter- The plates are then heated at 85 C.

TABLE I.ASSAY OF FIBRINOLYTIC AGENT BY THE FIBRIN PLATE METHOD Diameter(111.111.) of Clearing Time (min.) Amount of Agent/cc.

This table clearly shows that there is a linear relationship between theactivity and amount of fibrinolytic agent. It follows that the secondassay method is susceptible to quantitation. Therefore, this methodpermits quantitative determination of the fibrinolytic activity of amold filtrate in a reliable manner.

For the production of the new fibrinolytic agents active fungi, as theycan be selected and distinguished from inactive fungi, are cultivated onsuitable culture media. Culture media containing a substantial amount ofphosphates have a favorable effect on the amount of fibrinolytic agentproduced by the fungus. It is, of course, understood that the culturemedium contains a source of carbon, such as starches, dextrin, dextrose,lactose, sucrose, and other carbohydrates; a source of nitrogen such asammonium salts, nitrates, or organic nitrogen sources such as peptones,wheat bran; mineral salts which supply potassium, sodium, magnesium,nitrate, phosphate, sulfate ions; and trace elements, such as iron;vitamins, especially the vitamins of the B-group; and other mold growthpromoting substances. It has been found that the presence of largeamounts of phosphates increases the yield. Amounts of potassiummonophosphate of at least 1% and preferably of 1.2% to 1.5% of theculture medium have provide to be especially effective.

The following Table II clearly shows that a high phosphate content inthe culture medium has a favorable effect on the yield of fibrinolyticagent. In said table the amount of potassium monophosphate is given ing. per liter of culture medium. The pH given in said table is the pH ofthe culture filtrate after cultivation for 13 days at a temperature of25 C. The weight of the mycelium is given in g. after drying at 100 C.The last column shows the weight of the dried alcohol precipitate. It isclearly evident that the yield of precipitate decreases considerablywith the decrease in phosphate concentration, although the amount ofmycelium remains relatively constant. The activity of all fourprecipitated fibrinolytic agents on a dry weight'base is about the sameso that the increase in yield on cultivation with a large amount ofpotassium monophosphate represents a factual increase in the amount offibrinolytic agent produced.

TABLE II.-INFLUENCE OF PHOSPHATE CONTENT IN CULTURE MEDIUM ON YIELD OFFIBRINOLY'IIC AGENT pH of culture Weight of Weight of Amount of KH3PO4,g./l. medium mycelium dried alcohol after 13 days in g. precipitateachieved within 3 to 15 days depending upon cultivation temperature andother conditions.

The following Table III shows the effect of the cultivation temperatureupon yield and activity of the fibrinolytic agent produced. The samemold culture was cultivated at two difierent temperatures, namely at 30C. and at 37 C. The fibrinolytic agent was precipitated by the tannicacid method described hereinafter. The yield of precipitate isdetermined after 2, 4, 6 and 9 days. The activity of the precipitateswas determined according to the above mentioned secondmethod.Furthermore, the activity/yield coefiicient is determined by multiplyingthe activity with the yield.

TABLE III.-INFLUENCE F TEMPERATURE ON YIELD OF FIBRINOLYTIC AGENT ANDITS ACTIVITY This table clearly shows that at the higher cultivationtemperature of 37 C. the highest activity/yield coefficient is obtainedbetween the 4th and the 6th day while at the lower cultivation,temperature of 30 C. it takes at least six days to produce asatisfactory activity/yield coefiicient.

Cultivation can be carried out at a temperature between about 22 C. andabout 37 C. The preferred cultivation temperature is between about 28 C.and about 32 C. Substantially no activity was found on cultivation below20 C. and above 40 C. The fibrinolytic agents may be obtained by bothsurface or submerged cultivation.

The preferably inoculum is grown on a solid culture medium preferably onSabourauds dextrose medium, until abundant sporulation is observed. SuchWell sporulated inoculum is then transferred to the liquid culturemedium mentioned hereinabove. Three to four inoculums of the size ofabout 3 cm. in diametereach are transferred to a 5 1. bottle, containing1,000 cc. of said liquid culture medium. Of course, smaller or largervolumes of culture medium may also be used.

trations above 70%, cause a certain decrease in activity.

The alcohol precipitate contains the active agent. This Preferably suchan amount of tannic acid is added that Working up of the culture mediumin order to concentrate the fibrinolytic agent may be effected indifferent ways whereby after each step of isolation the fibrinolyticactivity of the resulting intermediate solution and products isdetermined in order to find out whether a specific step actually causesconcentration of the active agent.

One method of producing solutions of the fibrinolytic agents accordingto the present invention follows closely the method described fortesting molds for their fibrinolytic activity. According to saidprocess, the liquid culture medium which has a pH between 5.2 and 5.8 isfiltered or centrifuged to remove the mycelium. Preferably filtration iseffected through Whatman No. 2 paper. Two parts by volume of 95% ethanolprecooled, preferably to 20 C.,-are added to one part by volume of thefiltrate precooled to 010 C. The mixture is kept overnight in therefrigerator below 0 C. or is cooled to 20 C. and centrifuged afterstanding for half an hour. It was found that best results are obtainedwhen using two parts by volume of alcohol whereby the mixture has analcohol concentration between about 60% and about 65% Lower alcoholconcentrations'than about 40% do not cause precipitation. Higher alcoholconcentrations, i.e. concenthe concentration thereof in the filtratebegins with 25 mg. of tannic acid per cc. of culture filtrate andincreases with the amount of fibrinolytic agent in the bath. Tannic acidis preferably added in aqueous 10% solution (100 mg. per cc.). Tannicacid'precipitation is carried out at about 0 C. The resulting tannicacid precipitate is about 20 times as active as the alcohol precipitateon a dry weight base. Tannic acid, thus, has a greater selectiveprecipitating effect on the fibrinolytic agent than alcohol since onlyminimum amounts of salts are trapped in the precipitate.

The following Table IV shows the effect of varying amounts of tannicacid added to the mold filtrate. In order to carry out precipitationunder exactly the same conditions, the pI-I-value of the filtrate wasadjusted to a pH of 5.5 by the addition of 5 N sodium hydroxide solutionbefore the tannic acid was added. It is evident from this table thatthere is a relatively insignificant difference between the variousamounts of tannic acid used, provided that an amount of about 0.25 mg.of tannic acid per cc. of culture filtrate is used. The yield is givenin this table in mg. per 200 cc. of'filtrate. The activity is determinedaccording to the above mentioned second test method after min. and isexpressed in percent change in the diameter of the clearing as comparedwith the diameter at zero time of application of the test solution tothe plate. The activity/yield coefficient is expressed as the yieldmultiplied by the activity percent change. The table clearly shows thatbest results are achieved with amounts of about 0.75 mg. to 1.0 mg. oftannic acid per cc. of filtrate.

TABLE IV.YIELD 0F FIBRINOLYTIC AGENT IN RELA- TION TO THE AMOUNT OFTANNIC ACID ADDED TO THE CULTURE FILTRATE The tannic acid precipitate isobtained by centrifuging and decanting the supernatant liquid. Theprecipitate is washed with anhydrous acetone or any other tannic acidsolvent to remove the tannic acid. The remaining active precipitate isthen dried under vacuum. The resulting precipitates of active agent,whether obtained by tannic acid precipitation or by alcoholprecipitation may be further purified in order to remove insolublereactive material and contaminating soluble salts. Purification ispreferably elfected by resuspending the precipitate in water, separatingthe insoluble material, di-alyzing the solution against water,lyophilizing the dialyzed solution.

One unit of activity of the fibrinolytic agent is expressed as theamount of activity contained inone mg. of dry material which causesfragmentation of the fibrin plate prepared as described hereinabove, toa diameter of 1 cm. after 60 minutes'at 37 C., when applied to such aplate in an amount of 0.1' cc. of an 0.1% aqueous solution. Forinstance, the tannic acid precipitates correspond to 2 to 4 units/mg.

The resulting fibrinolytic agents, depending on their degree of purity,contain variable amounts of nitrogen. However, the conventional testsperformed for nitrogen, such as Ninhydrin, Micro-Kjeldahl associatedwith Nesslerization, and Tyrosin tests, have produced a typical colorreaction, and consequently, the total amount of nitrogen cannot bereliably reported.

Infrared spectra of relatively pure fibrinolytic substances have beenmade. The attached drawings show the following infrared spectra:

FIGURE 1 illustrates the infrared spectrum obtained from a dialyzed moldculture filtrate.

FIGURE 2 illustrates the infrared spectrum of a mold culture filtrateprecipitated with ethanol as described hereinabove and dialyzed.

FIGURE 3 illustrates the infrared spectrum from a mold culture filtrateprecipitated with tannic acid and dialyzed.

It will be noted from said spectra that the following bands are commonto the three preparations:

Near 3.0 microns, indicating the presence of an OH- group or an NH groupor a combination thereof.

Near 3.45 microns, indicating that the substance is an organic materialcontaining carbon and hydrogen.

Near 6.05 microns, indicating a carbon adjacent to a nitrogen.

Near 9.4- microns, indicating a carbon-oxygen bond, and from itsintensity, it would indicate a highly hydroxylated compound.

It will be noted that in the spectrum of FIGURE 3, an additional bandmay be clearly seen at 6.5 microns, indicating a secondary carbon tonitrogen vibration. However, it must be pointed out that this spectrumwas obtained with material utilizing tannic acid as a precipitant. Atthis wave length, aromatic compounds containing phenolic groups alsoabsorb, and such a phenolic group is present in tannic acid. Since thetannic acid precipitate has a much greater fibrinolytic activity, thisband may also be related to the fibrinolytic property of the product.

FIGURE 4 shows the ultraviolet spectrum for the same substance as wasused in FIGURE 3. It shows an absorption at 280 millimicrons which ischaracteristic of a phenolic group. Whether such a phenolic group is acomponent of the fibrinolytic agent itself or whether it is due totannic acid or the presence of a protein containing the phenolic tyrosinwhich also absorbs at 280 millimicrons, will have to be established.

By means of dialysis, utilizing a cellulose membrane with an averagepore size of 24 Angstroms, it is possible to further purify andconcentrate the active agent.

On suspending the dry alcohol precipitate in distilled water, thepH-value of the resulting supernatant solution is between 5.0 and 5.7 ata concentration of 1 mg. to mg./cc., while the tannic acid precipitatehas a pH between 5.9 and 6.1. The tannic acid precipitate forms withsmall amounts of water a viscous sticky paste. The active agent issoluble in water in amounts of 0.5 mg. to 5.0 mg. per cc. 10 mg. of theprecipitate are insoluble in ether, ethyl acetate, acetone, andgenerally in lipid solvents.

The precipitates were tested for their antigenic and pyrogenic elfectsand were found to be substantially free of such efiects.

' Experiments with dogs in' which a venous blood clot was produced,showed that intravenous injection of 0.250 mg. to 1.0 g. of the tannicacid-precipitated agents suspended in saline solutions caused graduallysis of the clot and reversal of the biochemical changes in the bloodclotting mechanisms which accompany the formation and propagation of theblood clot at the level of thrombosis. A very important additionalefiect of the new agent is its anti-inflammatory effect at the site ofthe clot. Furthermore, when an experimental clot is formed, it willordinarily continue to propagate, especially if there is an alterationof the endothelial lining. However, under treatment with thefibrinolytic agents according to the present invention, propagation doesnot continue, even when the dosage is inadequate to cause completedisappearance of the clot. It was also noted that, under treatment,bleeding occurred at the site of incision from small vessels which hadpreviously spontaneously thrombosed without ligatures.

The following examples serve to illustrate the present inventionwithout, however, limiting the same thereto.

Example 1.Preparati0n of an inoculum of Aspergillus oryzae strain No.B-1273 From a spore culture of Aspergillus oryzae strain No. -B1273 thesolid Sabourauds medium is streaked, using a 1 cm. loop and allowed tostand at 37 C. until the mycelium has developed and an abundant quantityof spores has been formed. Usually five to six days are required. A deepgreen color is noted and is an indication of satisfactory sporulationfor inoculation.

Example 2.--Cultivation of Aspergillus oryzae strain No. B-1273 Usingthe spores of the culture according to Example 1, three 2 cm. loops aretransferred, under sterile conditions, to 5 1. bottles, each containing1,000 cc. of the liquid culture medium referred to in col. 2 hereinaboveand having a pH of 4.5. Cultivation is effected at 29 C. untilsporulation sets in which is usually the case after a period of about 6days to 10 days. The pH is now about 5.4. Spo-rulation is readilyascertained by the occurrence of a yellowish or light brownish-greencolor on the surface of the mycelium.

Cultivation at 22-25 C. requires about 7 days to 14 days beforesporulation sets in, while cultivation at 34- 37 C. will result inbeginning sporulation after 3 days to 6 days.

Example 3.Precipitati0n of fibrinolytic agent by means of alcohol15.7 1. of the culture filtrate obtained on filtration of culturesprepared by cultivation at 25 C. for 7 days which are pre-cooled toabout 5 C., are precipitated by the addition of 31.5 1. of ethanol,pre-cooled to 20 C. The mixture is allowed to stand at about 18 C. to 20C. for 48 hours. The precipitate is filtered by suction at the same lowtemperature. The filtered precipitate is dried in a vacuum at 3740 C.for about 36 hours. About 74 g. of dry fibrinolytic agent are obtained.

Example 4.Precipitati0n of fibrinolytic agent by means of tannic acid14.5 1. of the mold culture filtrate obtained on filtration of culturesprepared by cultivation for 7 days at 25 C. and having a pH of 5.1,which are pre-cooled to about 5 C. are precipitated by the admixture of7.0 g. of tannic acid dissolved in cc. of water. The precipitate isallowed to completely settle out by standing at 4 C. for 48 hours. Asubstantial amount of the supernatant liquid is removed and theremainder, with the precipitate, is centrifuged. The centrifugedprecipitate is stirred with 12 cc. of anhydrous acetone at -2() C. 48cc. of acetone are admixed and the mixture is allowed to stand at -20 C.The precipitate is filtered on Whatman 50 paper, and washed with 30 cc.of acetone to remove the acetone soluble fractions. The Washedprecipitate is dried'in a vacuum. About 0.45 g. of dried fibrinolyticmaterial are obtained.

Example 5 .Purification of the alcohol precipitate The alcoholprecipitate obtained according to Example 3 is suspended in 740 cc. ofdistilled water while stirring.

9 The mixture is centrifuged and the supernatant liquid is adjusted to apH of 5.5 by the addition of5 N sodium hydroxide solution. To this isadded 1.5 mg. of tannic acid in finely divided form per cc. of saidsupernatant liquid, and the mixture is permitted to stand at C. for 24hours. The resulting precipitate is worked up in the same manner asdescribed hereinabove in Example 4. The precipitate is Washed withacetone as described in Example 4 and the washed material is dried atroom temperature in a vacuum. 0.280 g. of dried fibrinolytic agent areobtained.

Example 6 Example 7 Of the material obtained according to Example 280mg. are resu-spended in 56 cc. of distilled water and the resultingsuspension is worked up in the same manner as described in Example 6,resulting in a yield of about 12 mg. of highly fibrinolytic agent.

Of course, many changes and variations in the preparation of theinoculum, in the cultivation of the fibrinolytically active molds, inthe isolation of-the fibrinolytic agents from the mold culturefiltrates, in the purification of said agents, in their administrationin human and veterinary therapy, and the like may be made by thoseskilled in the art in accordance with the principles set forth hereinand in the claims annexed hereto.

We claim:

1. In a process of producing a fibrinolytic agent not substantiallyaffecting other blood and plasma constituents, the steps which comprisecultivating, on a liquid culture medium of a high phosphate ion content,a mold having fibrinolytic activity at a temperature between 22 C. and37 C. until sporulation sets in, said mold being selected from the groupconsisting of Aspergillus oryz ae B82i, Aspergillus oryzae Bl273,Aspergz'llus flavus B4m, and Absidia coerulea D-101, discontinuingcultivation, and separating the mycelium from the liquid culture mediumcontaining the active agent.

2. The process according to claim 1, wherein the liquid culture mediumcontains at least 1% of potassium monophosphate.

3. The process according to claim 1, wherein the pH- value of the liquidculture medium is initially about 4.4 to 4.5 and, at the timesporulation sets in, has increased to about 5.2 to 5.8.

4. In a process of producing a fibrinolytic agent not substantiallyafiecting other blood and plasma constituents, the steps which comprisecultivating, on a liquid culture medium of a high phosphate ion content,a mold having fibrinolytic activity at a temperature between about 22 C.and 37 C. until sporulation sets in, said mold being selected from thegroup consisting of Aspergillus oryzae B82i, Aspergillus oryzae Bl273,Asp'ergillus flavus B-4m, and Absidza coerulea D-lOl, separating themycelium from the liquid culture medium, adding alcohol to said liquidculture medium at a temperature not substantially exceeding 0 C. in anamount suflicient to produce an alcohol concentration of at least 60%,allowing the mixture to stand until precipitation is completed, andseparating the precipitate containing the fibrinolytic agent.

5. In a process of producing a fibrinolytic agent not substantiallyaffecting other blood and plasma constituents,

the steps which comprise cultivating, on a liquid culture medium of ahigh phosphate ion content, a mold having fibrinolytic activity at atemperature between 22 C. and 37 C. until sporulation sets in, said moldbeing selected from the group consisting of Aspergillus oryzae B82i,Aspergillus oryzae-B-1273, Aspergillus flavus B-4m, and Absz'a'iacoerulea D101, separating themycelium from the liquid culture medium,adding tannic acid to said liquid culture medium at about 0 C. in anamount sufilcient to cause substantially complete precipitation of thefibrinolytic agent, and separating the precipitate containing thefibrinolytic agent. i

6. In a process of producing a fibrinolytic agent not substantiallyaffecting other blood and plasma constituents, the steps which comprisecultivating, on a liquid culture medium of a high phosphate ion content,a mold having fibrinolytic activity at a temperature between 22 C. and37 C. until sporulation sets in, said mold being selected from the groupconsisting of Aspergillus oryzae B82i, Aspergillus oryzae Bl273,Aspergillus flavus B4m, and Absidia coerulea D-101, separating themycelium from the liquid culture medium, adding tannic acid to saidliquid culture medium at about 0 C. in an amount sufficient to causesubstantially complete precipitation of the fibrinolytic agent,separating the precipitate containing the fibrinolytic agent, stirringthe precipitate with anhydrous acetone, filtering the mixture, removingthe acetone, repeating said stirring with acetone, filtering, andremoving the acetone until substantially all the acetone solublematerial has been removed, drying the acetone-treated precipitate,suspending said precipitate in water, removing insoluble matter bycentrifuging, and dialyzing the supernatant liquid against running waterat about 10 C. until substantially all dialyzable material has beenremoved.

- 7. The fibrinolytic agent obtained from cultures of molds beingselected from the group consisting of Aspergillus oryzae B82i,Aspergillus oryzae Bl273, Aspergillus flavus B4m, and Absidia coeruleaD101, said agent not substantially aifecting other blood and plasmaconstituents, being soluble in water, insoluble in ether, ethyl acetate,and acetone, the pH-value of its aqueous solution being between 5.2 and6.1, aqueous solutions of said fibrinolytic agent losing theirfibrinolytic activity on adjusting their pH-value above a pH of 6.5 andbelow a pH of 3.0 when standing, the dry fibrinolytic agent beinginactivated on standing at a temperature of above 75 C., the infraredspectrum of the alcohol-precipitated fibrinolytic agent showingabsorption bands near 3 microns, near 3.45 microns, near 6.05 microns,and near 9.4' microns, and the infrared spectrum of the tannicacid-precipitated fibrinolytic agent showing absorption bands near 3microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, andnear 9.4 microns, the ultraviolet absorption spectrum of said tannicacid-precipitated fibrinolytic agent showing an absorption at 280millimicrons, the fibrinolytic agent not being dialyzable against Waterthrough a cellulose membrane with an average pore size of 24 Angstroms,the. agent being substantially non-pyrogenic and nonantigenic, itsaqueous solution causing lysis of thrombinclotted platelet-poor humanand bovine plasma and of thrombin-clotted human and bovine fibrinogenplates.

7 References Cited by the Examiner UNITED STATES PATENTS 2,716,084 8/1955 Carlson et al. 66 2,753,291 7/1956 Mowat et al 195-66 2,795,529 6/1957 Album et al. 16773 2,857,316 10/1958 Grimm 195-6 2,857,317 10/ 1958Grimm 195 6 FOREIGN PATENTS 566,369 11/ 1958 Canada.

(Other references on following page) 3,2 56,1 5 7 1 1 1 2 OTHERREFERENCES Yoshida et 21.: Free. of the Int. Sym. on Enzyme Chem,Australian Journal of Biological Sciences, vol. 6 (1195 3 Tokyo 1957504-509 pp. 410-462, pp. 411, 418, 428-432 and 447-451 par- LOUIS MONACELL, Primary Examinerticularly relied upon.

Proc. Int. Sym. Enzyme Chemistry, Tokyo, 1957, pp. 5 MORRIS WOLK,TOBIAS' LEVOW, ABRAHAM 482-485, 504 509 H. WINKELSTEIN, Exammers.

Stefani et 211.: Proceedings of the Society for Experi- F CACCIAPAGLIA,L, M, SHAPIRO, mental Biology and Medicine, vol. 99, pp. 504-507, 1958.Assistant Examiners,

1. IN A PROCESS OF PRODUCING A FIBRINOLYTIC AGENT NOT SUBSTANTIALLYAFFECTING OTHER BLOOD AND PLASMA CONSTITUENTS, THE STEPS WHICH COMPRISECULTIVATING, ON A LIQUID CULTURE MEDIUM OF A HIGH PHOSPHATE ION CONTENT,A MOLD HAVING FIBRINOLYTIC ACTIVITY AT A TEMPERATURE BETWEEN 22*C. AND37*C. UNTIL SPORULATION SETS IN, SAID MOLD BEING SELECTED FROM THE GROUPCONSISTING OF ASPERGILLUS ORZAE B-82I, ASPERGILLUS ORYZAE B-1273,ASPERGILLUS FLAVUS B-4M, AND ABSIDIA COERULEA D-101, DISCONTINUINGCULTIVATION, AND SEPARATING THE MYCELIUM FROM THE LIQUID CULTURE MEDIUMCONTAINING THE ACTIVE AGENT.
 7. THE FIBRINOLYTIC AGENT OBTAINED FROMCULTURES OF MOLDS BEING SELECTED FROM THE GROUP CONSISTING OFASPERGILLUS ORYZAE B-82I, ASPERGILLUS ORYZAE B-1273, ASPERGILLUS FLAVUSB-4M, AND ABSIDIA COERULEA D-101, SAID AGENT NOT SUBSTANTIALLY AFFECTINGOTHER BLOOD AND PLASMA CONSTITUENTS, BEING SOLUBLE IN WATER, INSOLUBLEIN ETHER, ETHYL ACETATE, AND ACETONE, THE PH-VALUE OF ITS AQUEOUSSOLUTION BEING BETWEEN 5.2 AND 6.1, AQUEOUS SOLUTIONS OF SAIDFIBRINOLYTIC AGENT LOSING THEIR FIBRINOLYTIC ACTIVITY ON ADJUSTING THEIRPH-VALUE ABOVE A PH OF 6.5 AND BELOW A PH OF 3.0 WHEN STANDING, THE DRYFIBRINOLYTIC AGENT BEING INACTIVIATED ON STANDING AT A TEMPERATURE OFABOVE 75*C., THE INFRARED SPECTRUM OF THE ALCOHOL-PRECIPITATEDFIBRINOLYTIC AGENT SHOWING ABSORPTION BANDS NEAR 3 MICRONS, NEAR 3.45MICRONS, NEAR 6.05 MICRONS, AND NEAR 9.4 MICRONS, AND THE INFRAREDSPECTRUM OF THE TANNIC ACID-PRECIPITATED FIBRINOLYTIC AGENT SHOWINGABSORPTION BANDS NEAR 3 MICRONS, NEAR 3.45 MICRONS, NEAR 6.05 MICRONS,NEAR 6.5 MICRONS, AND NEAR 9.4 MICRONS, THE ULTRAVIOLET ABSORPTIONSPECTRUM OF SAID TANNIC ACID-PRECIPITATED FIBRINOLYTIC AGNET SHOWING ANABSORPTION AT 280 MILLIMICRONS, THE FIBRINOLYTIC AGENT NOT BEINGDIALYZABLE AGAINST WATER THROUGH A CELLULOSE MEMBRANE WITH AN AVERAGEPORE SIZE OF 24 ANGSTROMS, THE AGENT BEING SUBSTANTIALLY NON-PYROGENICAND NONANTIGENIC, ITS AQUEOUS SOLUTION CAUSING LYSIS OF THROMBINCLOTTEDPLATELET-POOR HUMAN AND BOVINE PLASMA AND OF THROMBIN-CLOTTED HUMAN ANDBOVINE FIBRINOGEN PLATES.